Immunoassays for detecting an analyte in a sample are used extensively in various fields. Immunoassays can be used to detect analytes in various samples (such as saliva, blood, urine, serum, and sweat) to aid in diagnosis, prognosis and/or treatment of diseases. For instance, immunoassays are routinely performed for monitoring signs of early pregnancy, the presence of tumors, infectious diseases and drug abuse. Immunoassay is effected by the specific binding sites contained between immune molecules, such as antigen/antibody, hapten/antibody, and biotin/antibiotin. Most immunoassays are performed on a solid phase, such as on lateral-flow test strips, glass or plastic multi-porous discs, or immune chromatography devices.
There are two common formats of immunoassays, namely the sandwich assay and the competitive assay. In a competitive immunoassay, the analyte competes with a labeled analyte to bind with antibodies. This format has been commonly used to detect small analytes such as hapten in a sample. The label used can be one that indicates its presence as a color band in the detection or result reading zone. Reagents and devices utilizing competitive principle have been described in detail in U.S. Pat. Nos. 4,235,601, 4,442,204, 5,229,073. For devices utilizing competitive principles, if the color of the detection zone does not change or if no color band appears in detection zone or result reading zone, this indicates a positive result, and thus suggests that an analyte may be present in a sample. Conversely, if color is already present in the detection zone or result reading zone changes or if a color band appears, this can be interpreted as a negative result, which further suggests that the analyte may not be present in a sample.
Other devices in which a positive result is directly indicated have been described in, for example, U.S. Pat. Nos. 5,028,535, 5,089,391, 5,627,526, 5,143,852, 5,480,792, and 5,985,579. When such a device is used to detect, for example, hapten, if the analyte is present or if its concentration is higher than an anticipated threshold, the color appearing in the test zone suggests a positive result. Conversely, a negative result suggests analyte may not be present or that the analyte concentration is lower than an anticipated threshold. Although the above-mentioned devices and methods can detect several analytes at one time, there are still many disadvantages to the current devices available. For instance, samples and multiple antibodies for binding analytes have to be put in a sample well for about 10-15 minutes to react prior to be applied to the nitrocellulose (NC) membrane. Furthermore, the NC membrane has to be washed repetitively prior to reading the results. Thus such devices are time-consuming, inconvenient to operate, and require multi-steps reactions.
In U.S. Pat. No. 5,451,504 a test strip is disclosed comprising three specific zones, a mobilization zone, a trap zone and a detection zone. An antibody, bound to a color label in the mobilization zone, can bind with an analyte to form a complex (a color label-antibody-analyte complex). The complex can be moved by a liquid to a trap zone where a ligand is immobilized. The immobilized ligand is not able to bind to an antibody that has already formed a complex with an analyte. Such a complex is stable and cannot be affected by the immobilized ligand. The ligand can only bind an antibody that is freely floating and not already bound to an analyte. The analyte-antibody complex can then be captured by an immobilized detection receptor as to show a direct positive result. However, the device described in this patent requires that the affinity between the antibody and the analyte be higher than the affinity between the antibody and the ligand. In practice, it is very difficult to select the antibody with these limited features to apply to this invention.
In U.S. Pat. No. 6,699,722 a test strip is described consisting of a sample application zone, a mobilization zone, a first capture zone and a second capture zone. In this device, these zones are arranged along the pathway of liquid flowing, so that the liquid flows through each zone sequentially. The first capture zone is coated with a first capture antibody and the second capture zone is coated with a second capture antibody. The molecular weight of a labeled analyte analog is heavier than the weight of the analyte in the sample. If an analyte is present in the sample, the labeled analyte analog and analyte will move along following the pathway of the flowing liquid with the analyte traveling faster than the analyte analog. As result, the analyte will reach the first capture zone earlier than labeled analyte analog. The analyte is captured and fixed by the first capture antibody before the labeled analyte analog reaches the capture zone. The, labeled analyte analog will continue to flow past the first capture zone and come in contact with the second capture zone coated with a second capture antibody. The color in second capture zone will then change in response to the binding of the labeled analyte analogs further indicates a positive result. In practice, such a test strip is inconvenient and the results from such a test strip are not accurate.